The three-dimensional conformation of a protein molecule is determined by its amino acid sequence, and the details of a protein's conformation determine its chemistry.
A protein generally consists of a polypeptide backbone with attached side chains. The sequence of the chemically different side chains of the amino acid makes each protein distinct. The folded structure of a protein is stabilized by noncovalent interactions (e.g., hydrogen bonds, ionic bonds, and van der Waals attractions) that form between different parts of the polypeptide chain. one part of the chain and another. The stability of each folded shape is determined by the combined strength of large numbers of such noncovalent bonds.
Each protein has four levels of structural organization. The amino acid sequence is the primary structure of the protein. The secondary structure is defined by patterns of hydrogen bonds between backbone amide and carboxyl groups without consideration of sidechain-mainchain and sidechain-sidechain hydrogen bond (e.g. α helix, β-sheet). The tertiary structure, the full three-dimensional organization of a polypeptide chain, is the manner in which the sheets and helices of the secondary structure of a protein fold on themselves to define the three-dimensional structure. Quaternary structure refers to the complete structure of a protein molecule formed as a complex of more than one polypeptide chain.
Protein domains are structural units that fold more or less independently of each other to form globular compact structures. A domain usually contains between about 40 and about 350 amino acids, and is the modular unit from which many larger proteins are constructed. The different domains of a protein often are associated with different functions. The final folded structure, or conformation, adopted by any polypeptide chain generally is the one in which the free energy is minimized.
1. Kinases
Kinases are a ubiquitous group of enzymes that catalyze the phosphoryl transfer reaction from a phosphate donor (usually adenosine-5′-triphosphate (ATP)) to a receptor substrate. Although all kinases catalyze essentially the same phosphoryl transfer reaction, they display remarkable diversity in their substrate specificity, structure, and the pathways in which they participate. A recent classification of all available kinase sequences (approximately 60,000 sequences) indicates kinases can be grouped into 25 families of homologous proteins. These kinase families are further assembled into 12 fold groups based on similarity of structural fold. Further, 22 of the 25 families (approximately 98.8% of all sequences) belong to 10 fold groups for which the structural fold is known. Of the other 3 families, polyphosphate kinase forms a distinct fold group, and the 2 remaining families are both integral membrane kinases and comprise the final fold group. These fold groups not only include some of the most widely spread protein folds, such as Rossmann-like fold (three or more parallel beta strands linked by two alpha helices in the topological order beta-alpha-beta-alpha-beta), ferredoxin-like fold (a common α+β protein fold with a signature βαββαβ secondary structure along its backbone), TIM-barrel fold (meaning a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone), and antiparallel β-barrel fold (a beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last), but also all major classes (all α, all β, α+β, α/β) of protein structures. Within a fold group, the core of the nucleotide-binding domain of each family has the same architecture, and the topology of the protein core is either identical or related by circular permutation. Homology between the families within a fold group is not implied.
Group I (23,124 sequences) kinases incorporate protein SIT-Y kinase, atypical protein kinase, lipid kinase, and ATP grasp enzymes and further comprise the protein SIT-Y kinase, and atypical protein kinase family (22,074 sequences). These kinases include: choline kinase (EC 2.7.1.32); protein kinase (EC 2.7.137); phosphorylase kinase (EC 2.7.1.38); homoserine kinase (EC 2.7.1.39); I-phosphatidylinositol 4-kinase (EC 2.7.1.67); streptomycin 6-kinase (EC 2.7.1.72); ethanolamine kinase (EC 2.7.1.82); streptomycin 3′-kinase (EC 2.7.1.87); kanamycin kinase (EC 2.7.1.95); 5-methylthioribose kinase (EC 2.7.1.100); viomycin kinase (EC 2.7.1.103); [hydroxymethylglutaryl-CoA reductase (NADPH2)] kinase (EC 2.7.1.109); protein-tyrosine kinase (EC 2.7.1.112); [isocitrate dehydrogenase (NADP+)] kinase (EC 2.7.1.116); [myosin light-chain] kinase (EC 2.7.1.117); hygromycin-B kinase (EC 2.7.1.119); calcium/calmodulin-dependent protein kinase (EC 2.7.1.123); rhodopsin kinase (EC 2.7.1.125); [beta-adrenergic-receptor] kinase (EC 2.7.1.126); [myosin heavy-chain] kinase (EC 2.7.1.129); [Tau protein] kinase (EC 2.7.1.135); macrolide 2′-kinase (EC 2.7.1.136); I-phosphatidylinositol 3-kinase (EC 2.7.1.137); [RNA-polymerase]-subunit kinase (EC 2.7.1.141); phosphatidylinositol-4,5-bisphosphate 3-kinase (EC 2.7.1.153); and phosphatidylinositol-4-phosphate 3-kinase (EC 2.7.1.154). Group further comprises the lipid kinase family (321 sequences). These kinases include: I-phosphatidylinositol-4-phosphate 5-kinase (EC 2.7.1.68); I D-myo-inositol-triphosphate 3-kinase (EC 2.7.1.127); inositol-tetrakisphosphate 5-kinase (EC 2.7.1.140); I-phosphatidylinositol-5-phosphate 4-kinase (EC 2.7.1.149); I-phosphatidylinositol-3-phosphate 5-kinase (EC 2.7.1.150); inositol-polyphosphate multikinase (EC 2.7.1.151); and inositol-hexakiphosphate kinase (EC 2.7.4.21). Group I further comprises the ATP-grasp kinases (729 sequences) which include inositol-tetrakisphosphate I-kinase (EC 2.7.1.134); pyruvate, phosphate dikinase (EC 2.7.9.1); and pyruvate, water dikinase (EC 2.7.9.2).
Group II (17,071 sequences) kinases incorporate the Rossman-like kinases. Group II comprises the P-loop kinase family (7,732 sequences). These include gluconokinase (EC 2.7.1.12); phosphoribulokinase (EC 2.7.1.19); thymidine kinase (EC 2.7.1.21); ribosylnicotinamide kinase (EC 2.7.1.22); dephospho-CoA kinase (EC 2.7.1.24); adenylylsulfate kinase (EC 2.7.1.25); pantothenate kinase (EC 2.7.1.33); protein kinase (bacterial) (EC 2.7.1.37); uridine kinase (EC 2.7.1.48); shikimate kinase (EC 2.7.1.71); deoxycytidine kinase (EC 2.7.1.74); deoxyadenosine kinase (EC 2.7.1.76); polynucleotide 5′-hydroxyl-kinase (EC 2.7.1.78); 6-phosphofructo-2-kinase (EC 2.7.1.105); deoxyguanosine kinase (EC 2.7.1.113); tetraacyldisaccharide 4′-kinase (EC 2.7.1.130); deoxynucleoside kinase (EC 2.7.1.145); adenosylcobinamide kinase (EC 2.7.1.156); polyphosphate kinase (EC 2.7.4.1); phosphomevalonate kinase (EC 2.7.4.2); adenylate kinase (EC 2.7.4.3); nucleoside-phosphate kinase (EC 2.7.4.4); guanylate kinase (EC 2.7.4.8); thymidylate kinase (EC 2.7.4.9); nucleoside-triphosphate-adenylate kinase (EC 2.7.4.10); (deoxy)nucleoside-phosphate kinase (EC 2.7.4.13); cytidylate kinase (EC 2.7.4.14); and uridylate kinase (EC 2.7.4.-). Group II further comprises the phosphoenolpyruvate carboxykinase family (815 sequences). These enzymes include protein kinase (HPr kinase/phosphatase) (EC 2.7.1.37); phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32); and phosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49). Group II further comprises the phosphoglycerate kinase (1,351 sequences) family. These enzymes include phosphoglycerate kinase (EC 2.7.2.3) and phosphoglycerate kinase (GTP) (EC 2.7.2.10). Group II further comprises the aspartokinase family (2,171 sequences). These enzymes include carbamate kinase (EC 2.7.2.2); aspartate kinase (EC 2.7.2.4); acetylglutamate kinase (EC 2.7.2.8 1); glutamate 5-kinase (EC 2.7.2.1) and uridylate kinase (EC 2.7.4.-). Group II further comprises the phosphofructokinase-like kinase family (1,998 sequences). These enzymes include 6-phosphofructokinase (EC 2.7.1.1 1); NAD(+) kinase (EC 2.7.1.23); I-phosphofructokinase (EC 2.7.1.56); diphosphate-fructose-6-phosphate I-phosphotransferase (EC 2.7.1.90); sphinganine kinase (EC 2.7.1.91); diacylglycerol kinase (EC 2.7.1.107); and ceramide kinase (EC 2.7.1.138). Group II further comprises the ribokinase-like family (2,722 sequences). These enzymes include: glucokinase (EC 2.7.1.2); ketohexokinase (EC 2.7.1.3); fructokinase (EC 2.7.1.4); 6-phosphofructokinase (EC 2.7.1.11); ribokinase (EC 2.7.1.15); adenosine kinase (EC 2.7.1.20); pyridoxal kinase (EC 2.7.1.35); 2-dehydro-3-deoxygluconokinase (EC 2.7.1.45); hydroxymethylpyrimidine kinase (EC 2.7.1.49); hydroxyethylthiazole kinase (EC 2.7.1.50); I-phosphofructokinase (EC 2.7.1.56); inosine kinase (EC 2.7.1.73); 5-dehydro-2-deoxygluconokinase (EC 2.7.1.92); tagatose-6-phosphate kinase (EC 2.7.1.144); ADP-dependent phosphofructokinase (EC 2.7.1.146); ADP-dependent glucokinase (EC 2.7.1.147); and phosphomethylpyrimidine kinase (EC 2.7.4.7). Group II further comprises the thiamin pyrophosphokinase family (175 sequences) which includes thiamin pyrophosphokinase (EC 2.7.6.2). Group II further comprises the glycerate kinase family (107 sequences) which includes glycerate kinase ((EC 2.7.1.31).
Group III kinases (10,973 sequences) comprise the ferredoxin-like fold kinases. Group III further comprises the nucleoside-diphosphate kinase family (923 sequences). These enzymes include nucleoside-diphosphate kinase (EC 2.7.4.6). Group III further comprises the HPPK kinase family (609 sequences). These enzymes include 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2.7.6.3). Group III further comprises the guanido kinase family (324 sequences). These enzymes include guanidoacetate kinase (EC 2.7.3.1); creatine kinase (EC 2.7.3.2); arginine kinase (EC 2.7.3.3); and lombricine kinase (EC 2.7.3.5). Group III further comprises the histidine kinase family (9,117 sequences). These enzymes include protein kinase (histidine kinase) (EC 2.7.1.37); [pyruvate dehydrogenase (lipoamide)] kinase (EC 2.7.1.99); and [3-methyl-2-oxybutanoate dehydrogenase(lipoamide)] kinase (EC 2.7.1.115).
Group IV kinases (2,768 sequences) incorporate ribonuclease H-like kinases. These enzymes include hexokinase (EC 2.7.1.1); glucokinase (EC 2.7.1.2); fructokinase (EC 2.7.1.4); rhamnulokinase (EC 2.7.1.5); mannokinase (EC 2.7.1.7); gluconokinase (EC 2.7.1.12); L-ribulokinase (EC 2.7.1.16); xylulokinase (EC 2.7.1.17); erythritol kinase (EC 2.7.1.27); glycerol kinase (EC 2.7.1.30); pantothenate kinase (EC 2.7.1.33); D-ribulokinase (EC 2.7.1.47); L-fucolokinase (EC 2.7.1.51); L-xylulokinase (EC 2.7.1.53); allose kinase (EC 2.7.1.55); 2-dehydro-3-deoxygalactonokinase (EC 2.7.1.58); N-acetylglucosamine kinase (EC 2.7.1.59); N-acylmannosamine kinase (EC 2.7.1.60); polyphosphate-glucose phosphotransferase (EC 2.7.1.63); beta-glucoside kinase (EC 2.7.1.85); acetate kinase (EC 2.7.2.1); butyrate kinase (EC 2.7.2.7); branched-chain-fatty-acid kinase (EC 2.7.2.14); and propionate kinase (EC 2.7.2.-).
Group V kinases (1,119 sequences) incorporate TIM β/α-barrel kinases. These enzymes include pyruvate kinase (EC 2.7.1.40).
Group VI kinases (885 sequences) incorporate GHMP kinases. These enzymes include galactokinase (EC 2.7.1.6); mevalonate kinase (EC 2.7.1.36); homoserine kinase (EC 2.7.1.39); L-arabinokinase (EC 2.7.1.46); fucokinase (EC 2.7.1.52); shikimate kinase (EC 2.7.1.71); 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythriol kinase (EC 2.7.1.148); and phosphomevalonate kinase (EC 2.7.4.2)
Group VII kinases (1,843 sequences) incorporate AIR synthetase-like kinases. These enzymes include thiamine-phosphate kinase (EC 2.7.4.16) and selenide, water dikinase (EC 2.7.9.3).
Group VIII kinases (565 sequences) incorporate riboflavin kinases (565 sequences). These enzymes include riboflavin kinase (EC 2.7.1.26).
Group IX kinases (197 sequences) incorporate dihydroxyacetone kinases. These enzymes include glycerone kinase (EC 2.7.1.29).
Group X kinases (148 sequences) incorporate putative glycerate kinases. These enzymes include glycerate kinase (EC 2.7.1.31).
Group XI kinases (446 sequences) incorporate polyphosphate kinases. These enzymes include polyphosphate kinases (EC 2.7.4.1).
Group XII kinases (263 sequences) incorporate integral membrane kinases, Group XII comprises the dolichol kinase family. These enzymes include dolichol kinases (EC 2.7.1.108), Group XII further comprises the undecaprenol kinase family. These enzymes include undecaprenol kinases (EC 2.7.1.66).
Kinases play indispensable roles in numerous cellular metabolic and signaling pathways, and they are among the best-studied enzymes at the structural, biochemical, and cellular levels. Despite the fact that all kinases use the same phosphate donor (in most cases, ATP) and catalyze apparently the same phosphoryl transfer reaction, they display remarkable diversity in their structural folds and substrate recognition mechanisms. This is probably due largely to the extraordinary diverse nature of the structures and properties of their substrates.
2. Mitogen-activated Protein Kinase-activated Protein Kinases (MK2 and MK3)
Different groups of MAPK-activated protein kinases (MAP-KAPKs) have been defined downstream of mitogen-activated protein kinases (MAPKs). These enzymes transduce signals to target proteins that are not direct substrates of the MAPKs and, therefore, serve to relay phosphorylation-dependent signaling with MAPK cascades to diverse cellular functions. One of these groups is formed by the three MAPKAPKs: MK2, MK3 (also known as 3 pK), and MK5 (also designated PRAK). Mitogen-activated protein kinase-activated protein kinase 2 (also referred to as “MAPKAPK2”, “MAPKAP-K2”, “MK2”) is a kinase of the serine/threonine (Ser/Thr) protein kinase family. MK2 is highly homologous to MK3 (approximately 75% amino acid identity). The kinase domains of MK2 and MK3 are most similar (approximately 35% to 40% identity) to calcium/calmodulin-dependent protein kinase (CaMK), phosphorylase b kinase, and the C-terminal kinase domain (CTKD) of the ribosomal S6 kinase (RSK) isoforms. The mk2 gene encodes two alternatively spliced transcripts of 370 amino acids (MK2A) and 400 amino acids (MK2B). The mk3 gene encodes one transcript of 382 amino acids. The MK2-and MK3 proteins are highly homologous, yet MK2A possesses a shorter C-terminal region. The C-terminus of MK2B contains a functional bipartite nuclear localization sequence (NLS) (Lys-Lys-Xaa10-Lys-Arg-Arg-Lys-Lys) [SEQ ID NO: 45]that is not present in the shorter MK2A isoform, indicating that alternative splicing determines the cellular localization of the MK2 isoforms. MK3 possesses a similar nuclear localization sequence. The nuclear localization sequence found in both MK2B and MK3 encompasses a D domain (Leu-Leu-Lys-Arg-Arg-Lys-Lys) [SEQ ID NO: 46]that studies have shown to mediate the specific interaction of MK2B and MK3 with p38α, and p38β. MK2B and MK3 also possess a functional nuclear export signal (NES) located N-terminal to the NLS and D domain. The NES in MK2B is sufficient to trigger nuclear export following stimulation, a process which may be inhibited by leptomycin B. The sequence N-terminal to the catalytic domain in MK2 and MK3 is proline rich and contains one (MK3) or two (MK2) putative Src homology 3 (SH3) domain-binding sites, which studies have shown, for MK2, to mediate binding to the SH3 domain of c-Abl in vitro. Recent studies suggest that this domain is involved in MK2-mediated cell migration.
MK2B and MK3 are located predominantly in the nucleus of quiescent cells while MK2A is present in the cytoplasm. Both MK2B and MK3 are rapidly exported to the cytoplasm via a chromosome region maintenance protein (CRM1)-dependent mechanism upon stress stimulation. Nuclear export of MK2B appears to be mediated by kinase activation, as phosphomimetic mutation of Thr334 within the activation loop of the kinase enhances the cytoplasmic localization of MK2B. Without being limited by theory, it is thought that MK2B and MK3 may contain a constitutively active NLS and a phosphorylation-regulated NES.
MK2 and MK3 appear to be expressed ubiquitously, with predominant expression in the heart, in skeletal muscle, and in kidney tissues.
2.1. Activation
Various activators of p38α and p38β potently stimulate MK2 and MK3 activity. p38 mediates the in vitro and in vivo phosphorylation of MK2 on four praline-directed sites: Thr25, Thr222, Ser272, and Thr334. Of these sites, only Thr25 is not conserved in MK3. Without being limited by theory, while the function of phosphorylated Thr25 in unknown, its location between the two SH3 domain-binding sites suggests that it may regulate protein-protein interactions. Thr222 in MK2 (Thr201 in MK3) is located in the activation loop of the kinase domain and has been shown to be essential for MK2 and MK3 kinase activity. Thr334 in MK2 (Thr313 in MK3) is located C-terminal to the catalytic domain and is essential for kinase activity. The crystal structure of MK2 has been resolved and, without being limited by theory, suggests that Thr334 phosphorylation may serve as a switch for MK2 nuclear import and export. Phosphorylation of Thr334 also may weaken or interrupt binding of the C terminus of MK2 to the catalytic domain, exposing the NES and promoting nuclear export.
Studies have shown that, while p38 is capable of activating MK2 and MK3 in the nucleus, experimental evidence suggests that activation and nuclear export of MK2 and MK3 are coupled by a phosphorylation-dependent conformational switch that also dictates p38 stabilization and localization, and the cellular location of p38 itself is controlled by MK2 and possibly MK3. Additional studies have shown that nuclear p38 is exported to the cytoplasm in a complex with MK2 following phosphorylation and activation of MK2. The interaction between p38 and MK2 may be important for p38 stabilization since studies indicate that p38 levels are low in MK2-deficient cells and expression of a catalytically inactive MK2 protein restores p38 levels.
2.2. Substrates and Functions
MK2 shares many substrates with MK3. Both enzymes have comparable substrate preferences and phosphorylate peptide substrates with similar kinetic constants. The minimum sequence required for efficient phosphorylation by MK2 was found to be Hyd-Xaa-Arg-Xaa-Xaa-pSer/Thr, where Hyd is a bulky hydrophobic residue.
Experimental evidence supports a role for p38 in the regulation of cytokine biosynthesis and cell migration. The targeted deletion of the mk2 gene in mice suggested that although p38 mediates the activation of many similar kinases, MK2 seems to be the key kinase responsible for these p38-dependent biological processes. Loss of MK2 leads (i) to a defect in lipopolysaccharide (LPS)-induced synthesis of cytokines such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and gamma interferon (IFN-γ) and (ii) to changes in the migration of mouse embryonic fibroblasts, smooth muscle cells, and neutrophils. Consistent with a role for MK2 in inflammatory responses, MK2-deficient mice show increased susceptibility to Listeria monocytogenes infection and reduced inflammation-mediated neuronal death following focal ischemia. Since the levels of p38 protein also are reduced significantly in MK2-deficient cells, it was necessary to distinguish whether these phenotypes were due solely to the loss of MK2. To achieve this, MK2 mutants were expressed in MK2-deficient cells, and the results indicated that the catalytic activity of MK2 was not necessary to restore p38 levels but was required to regulate cytokine biosynthesis.
2.3. Regulation of mRNA Translation.
Studies have shown that MK2 increases TNFα production by increasing the rate of translation of its mRNA; no significant reductions in the transcription, processing, and shedding of TNFα could be detected in MK2-deficient mice. The p38 pathway is known to play an important role in regulating mRNA stability, and MK2 represents a likely target by which p38 mediates this function. Studies utilizing MK2-deficient mice indicated that the catalytic activity of MK2 is necessary for its effects on cytokine production and migration, suggesting that, without being limited by theory, MK2 phosphorylates targets involved in mRNA stability. Consistent with this, MK2 has been shown to bind and/or phosphorylate the heterogeneous nuclear ribonucleoprotein (hnRNP) AO, tristetraprolin, the poly(A)-binding protein PABP1, and HuR (a ubiquitously expressed member of the elav (embryonic-lethal abnormal visual in Drospholia melanogaster) family of RNA-binding protein). These substrates are known to bind or copurify with mRNAs that contain AU-rich elements in the 3′ untranslated region, suggesting that MK2 may regulate the stability of AU-rich mRNAs such as TNFα. It currently is unknown whether MK3 plays similar functions, but LPS treatment of MK2-deficient fibroblasts completely abolished hnRNP AO phosphorylation, suggesting that MK3 is not able to compensate for the loss of MK2.
MK3 participates with MK2 in phosphorylation of the eukaryotic elongation factor 2 (eEF2) kinase. eEF2 kinase phosphorylates and inactivates eEF2. eEF2 activity is critical for the elongation of mRNA during translation, and phosphorylation of eEF2 on Thr56 results in the termination of mRNA translation. MK2 and MK3 phosphorylation of eEF2 kinase on Ser377 suggests that these enzymes may modulate eEF2 kinase activity and thereby regulate mRNA translation elongation.
2.4. Transcriptional Regulation by MK2 and MK3.
Nuclear MK2, similar to many MKs, contributes to the phosphorylation of cAMP response element binding (CREB), serum response factor (SRF), and transcription factor ER81. Comparison of wild-type and MK2-deficient cells revealed that MK2 is the major SRF kinase induced by stress, suggesting a role for MK2 in the stress-mediated immediate-early response. Both MK2 and MK3 interact with basic helix-loop-helix transcription factor E47 in vivo and phosphorylate E47 in vitro. MK2-mediated phosphorylation of E47 was found to repress the transcriptional activity of E47 and thereby inhibit E47-dependent gene expression, suggesting that MK2 and MK3 may regulate tissue-specific gene expression and cell differentiation.
2.5. Other Targets of MK2 and MK3.
Several other MK2 and MK3 substrates also have been identified, reflective of the diverse functions of MK2 and MK3 in several biological processes. The scaffolding protein 14-3-3ζ is a physiological MK2 substrate. Studies indicate 14-3-3ζ interacts with a number of components of cell signaling pathways, including protein kinases, phosphatases, and transcription factors. Additional studies have shown that MK2-mediated phosphorylation of 14-3-3ζ on Ser58 compromises its binding activity, suggesting that MK2 may affect the regulation of several signaling molecules normally regulated by 14-3-3ζ.
Additional studies have shown that MK2 also interacts with and phosphorylates the p16 subunit of the seven-member Arp2 and Arp3 complex (p16-Arc) on Ser77. p16-Arc has roles in regulating the actin cytoskeleton, suggesting that MK2 may be involved in this process. Further studies have shown that the small heat shock protein HSP27, lymphocyte-specific protein LSP-1, and vimentin are phosphorylated by MK2. HSP27 is of particular interest because it forms large oligomers which may act as molecular chaperones and protect cells from heat shock and oxidative stress. Upon phosphorylation, HSP27 loses its ability to form large oligomers and is unable to block actin polymerization, suggesting that MK2-mediated phosphorylation of HSP27 serves a homeostatic function aimed at regulating actin dynamics that would otherwise be destabilized during stress. MK3 also was shown to phosphorylate HSP27 in vitro and in vivo, but its role during stressful conditions has not yet been elucidated.
MK2 and MK3 also may phosphorylate 5-lipoxygenase. 5-lipoxygenase catalyzes the initial steps in the formation of the inflammatory mediators leukotrienes. Tyrosine hydroxylase, glycogen synthase, and Akt also were shown to be phosphorylated by MK2. Finally, MK2 phosphorylates the tumor suppressor protein tuberin on Ser1210, creating a docking site for 14-3-3. Tuberin and hamartin normally form a functional complex that negatively regulates cell growth by antagonizing mTOR-dependent signaling, suggesting that p38-mediated activation of MK2 may regulate cell growth by increasing 14-3-3 binding to tuberin.
3. Kinase Inhibition
The eukaryotic protein kinases constitute one of the largest superfamilies of homologous proteins that are related by virtue of their catalytic domains. Most related protein kinases are specific for either serine/threonine or tyrosine phosphorylation. Protein kinases play an integral role in the cellular response to extracellular stimuli. Thus, stimulation of protein kinases is considered to be one of the most common activation mechanisms in signal transduction systems. Many substrates are known to undergo phosphorylation by multiple protein kinases. A considerable amount of information on primary sequence of the catalytic domains of various protein kinases has been published. These sequences share a large number of residues involved in ATP binding, catalysis, and maintenance of structural integrity. Most protein kinases possess a well conserved 30-32 kDa catalytic domain.
Studies have attempted to identify and utilize regulatory elements of protein kinases. These regulatory elements include inhibitors, antibodies, and blocking peptides.
3.1. Inhibitors
Enzyme inhibitors are molecules that bind to enzymes thereby decreasing enzyme activity. The binding of an inhibitor may stop substrate from entering the active site of the enzyme and/or hinder the enzyme from catalyzing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically (e.g., by modifying key amino acid residues needed for enzymatic activity) so that it no longer is capable of catalyzing its reaction. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.
Enzyme inhibitors often are evaluated by their specificity and potency. The term “specificity” as used in this context rein refers to the selective attachment of an inhibitor or its lack of binding to other proteins. The term “potency” as used herein refers to an inhibitor's dissociation constant, which indicates the concentration of inhibitor needed to inhibit an enzyme.
Inhibitors of protein kinases have been studied for use as a tool in protein kinase activity regulation. Inhibitors have been studied for use with, for example, cyclin-dependent (Cdk) kinase, MAP kinase, serine/threonine kinase, Src Family protein tyrosine kinase, tyrosine kinase, calmodulin (CaM) kinase, casein kinase, checkpoint kinase (Chk1), glycogen synthase kinase 3 (GSK-3), c-Jun N-terminal kinase (INK), mitogen-activated protein kinase 1 (MEK), myosin light chain kinase (MLCK), protein kinase A, Akt (protein kinase B), protein kinase C, protein kinase G, protein tyrosine kinase, Raf kinase, and Rho kinase.
3.2. Antibodies
Antibodies are serum proteins the molecules of which possess small areas of their surface that are complementary to small chemical groupings on their targets. These complementary regions (referred to as the antibody combining sites or antigen binding sites) of which there are at least two per antibody molecule, and in some types of antibody molecules ten, eight, or in some species as many as 12, may react with their corresponding complementary region on the antigen (the antigenic determinant or epitope) to link several molecules of multivalent antigen together to form a lattice.
The basic structural unit of a whole antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids). The two heavy chains and two light chains are held together by a combination of noncovalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of a light chain and the N-terminal region of a heavy chain. Both light and heavy chains usually cooperate to form the antigen binding surface.
Human antibodies show two kinds of light chains, κ and λ; individual molecules of immunoglobulin generally are only one or the other. In normal serum, 60% of the molecules have been found to have κ determinants and 30 percent λ. Many other species have been found to show two kinds of light chains, but their proportions vary. For example, in the mouse and rat, λ chains comprise but a few percent of the total; in the dog and cat, κ chains are very low; the horse does not appear to have any κ chain; rabbits may have 5 to 40% λ, depending on strain and b-locus allotype; and chicken light chains are more homologous to λ than κ.
In mammals, there are five classes of antibodies, IgA, IgD, IgE, IgG, and IgM, each with its own class of heavy chain—α (for IgA), δ (for IgD), ε (for IgE), γ (for IgG) and μ (for IgM). In addition, there are four subclasses of IgG immunoglobulins (IgG1, IgG2, IgG3, IgG4) having γ1, γ2, γ3, and γ4 heavy chains respectively. In its secreted form, IgM is a pentamer composed of five four-chain units, giving it a total of 10 antigen binding sites. Each pentamer contains one copy of a J chain, which is covalently inserted between two adjacent tail regions.
All five immunoglobulin classes differ from other serum proteins in that they show a broad range of electrophoretic mobility and are not homogeneous. This heterogeneity—that individual IgG molecules, for example, differ from one another in net charge—is an intrinsic property of the immunoglobulins.
Monoclonal antibodies (mAbs) can be generated by fusing mouse spleen cells from an immunized donor with a mouse myeloma cell line to yield established mouse hybridoma clones that grow in selective media. A hybridoma cell is an immortalized hybrid cell resulting from the in vitro fusion of an antibody-secreting B cell with a myeloma cell. In vitro immunization, which refers to primary activation of antigen-specific B cells in culture, is another well-established means of producing mouse monoclonal antibodies.
Diverse libraries of immunoglobulin heavy (VH) and light (Vκ and Vλ) chain variable genes from peripheral blood lymphocytes also can be amplified by polymerase chain reaction (PCR) amplification. Genes encoding single polypeptide chains in which the heavy and light chain variable domains are linked by a polypeptide spacer (single chain Fv or scFv) can be made by randomly combining heavy and light chain V-genes using PCR. A combinatorial library then can be cloned for display on the surface of filamentous bacteriophage by fusion to a minor coat protein at the tip of the phage.
The technique of guided selection is based on human immunoglobulin V gene shuffling with rodent immunoglobulin V genes. The method entails (i) shuffling a repertoire of human λ light chains with the heavy chain variable region (VH) domain of a mouse monoclonal antibody reactive with an antigen of interest; (ii) selecting half-human Fabs on that antigen (iii) using the selected λ light chain genes as “docking domains” for a library of human heavy chains in a second shuffle to isolate clone Fab fragments having human light chain genes; (v) transfecting mouse myeloma cells by electroporation with mammalian cell expression vectors containing the genes; and (vi) expressing the V genes of the Fab reactive with the antigen as a complete IgG1, λ antibody molecule in the mouse myeloma.
As used herein, the term “antibody” includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term “antibody” includes polyclonal antibodies and monoclonal antibodies, and fragments thereof. Furthermore, the term “antibody” includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.
An antigenic determinant or epitope is an antigenic site on a molecule. Sequential antigenic determinants/epitopes essentially are linear chains. In ordered structures, such as helical polymers or proteins, the antigenic determinants/epitopes essentially would be limited regions or patches in or on the surface of the structure involving amino acid side chains from different portions of the molecule which could come close to one another. These are conformational determinants. As used herein, an epitope may be an antigenic determinant/antigen binding site on a kinase inhibiting peptide. The epitope may be primary, secondary, or tertiary-sequence related.
The principle of complementarity, which often is compared to the fitting of a key in a lock, involves relatively weak binding forces (hydrophobic and hydrogen bonds, van der Waals forces, and ionic interactions), which are able to act effectively only when the two reacting molecules can approach very closely to each other and indeed so closely that the projecting constituent atoms or groups of atoms of one molecule can fit into complementary depressions or recesses in the other. Antigen-antibody interactions show a high degree of specificity, which is manifest at many levels. Brought down to the molecular level, specificity means that the combining sites of antibodies to an antigen have a complementarity not at all similar to the antigenic determinants of an unrelated antigen. Whenever antigenic determinants of two different antigens have some structural similarity, some degree of fitting of one determinant into the combining site of some antibodies to the other may occur, and that this phenomenon gives rise to cross-reactions. Cross reactions are of major importance in understanding the complementarity or specificity of antigen-antibody reactions. Immunological specificity or complementarity makes possible the detection of small amounts of impurities/contaminations among antigens
The specificity of the interactions between certain antibodies and protein kinases has been studied for use in protein kinase activity regulation. Antibodies have been isolated for use with, for example, MAP kinase pathways, protein kinase A, protein kinase B, protein kinase G, serine/threonine kinases, glycogen-synthase kinase-3 (GSK-3), stress-activated protein (SAP) kinase pathways, and tyrosine kinases. Additionally, antibodies have been isolated for use with protein kinase inhibitors and protein kinase substrates.
3.3. Blocking Peptides
A peptide is a chemical compound that is composed of a chain of two or more amino acids whereby the carboxyl group of one amino acid in the chain is linked to the amino group of the other via a peptide bond. Peptides have been used inter alia in the study of protein structure and function. Synthetic peptides may be used inter alia as probes to see where protein-peptide interactions occur. Inhibitory peptides may be used inter alia in clinical research to examine the effects of peptides on the inhibition of protein kinases, cancer proteins and other disorders.
The use of several blocking peptides has been studied. For example, extracellular signal-regulated kinase (ERK), a MAPK protein kinase, is essential for cellular proliferation and differentiation. The activation of MAPKs requires a cascade mechanism whereby MAPK is phosphorylated by an upstream MAPKK (MEK) which is then, in turn phosphorylated by a third kinase MAPKKK (MEKK). The ERK inhibitory peptide functions as a MEK decoy by binding to ERK. It contains the amino-terminal 13 amino acids (GMPKKKPTPIQLN) [SEQ ID NO: 1] of MEK1 and binds to ERK. This blocks ERK activation by MEK as ERK is unable to interact with MEK. The ERK inhibitory peptide also contains a protein transduction (PTD) sequence (DRQIKIWFQNRRMKWKK) [SEQ ID NO: 2] derived from Antennapedia which renders the peptide cell permeable.
Other blocking peptides include autocamtide-2 related inhibitory peptide (AIP). This synthetic peptide is a highly specific and potent inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII). AIP is a non-phosphorylatable analog of autocamtide-2, a highly selective peptide substrate for CaMKII. AIP inhibits CaMKII with an IC50 of 100 nM (IC50 is the concentration of an inhibitor required to obtain 50% inhibition). The AIP inhibition is non-competitive with respect to syntide-2 (CaMKII peptide substrate) and ATP but competitive with respect to autocamtide-2. The inhibition is unaffected by the presence or absence of Ca2+/calmodulin. CaMKII activity is inhibited completely by AIP (1 μM) while PKA, PKC and CaMKIV are not affected. The amino acid sequence of AIP is: KKALRRQEAVDAL (Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Ala-Val-Asp-Ala-Leu) [SEQ ID NO: 3].
Other blocking peptides include cell division protein kinase 5 (Cdk5) inhibitory peptide (CIP). Cdk5 phosphorylates the microtubule protein tau at Alzheimer's Disease-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to amyloid β (A↓) peptides. Upon neuronal infections with CIP, CIPs selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. The reasons for the specificity demonstrated by CIP are not fully understood.
Additional blocking peptides have been studied for extracellular-regulated kinase 2 (ERK2), ERK3, p38/HOG1, protein kinase C, casein kinase II, Ca2+/calmodulin kinase IV, casein kinase II, Cdk4, Cdk5, DNA-dependent protein kinase (DNA-PK), serine/threonine-protein kinase PAK3, phosphoinositide (PI)-3 kinase, PI-5 kinase, PSTAIRE (the cdk highly conserved sequence), ribosomal S6 kinase, GSK-4, germinal center kinase (GCK), SAPK (stress-activated protein kinase), SEK1 (stress signaling kinase), and focal adhesion kinase (FAK).
3.4. Protein Transduction Domains
Protein transduction domains (PTDs) are a class of peptides capable of penetrating the plasma membrane of mammalian cells and of transporting compounds of many types and molecular weights across the membrane. These compounds include effector molecules, such as proteins, DNA, conjugated peptides, oligonucleotides, and small particles such as liposomes. When PTDs are chemically linked or fused to other proteins, the resulting fusion proteins still are able to enter cells. Although the exact mechanism of transduction is unknown, internalization of these proteins is not believed to be receptor-mediated or transporter-mediated. PTDs are generally 10-16 amino acids in length and may be grouped according to their composition, such as, for example, peptides rich in arginine and/or lysine.
The use of PTDs capable of transporting effector molecules into cells has become increasingly attractive in the design of drugs as they promote the cellular uptake of cargo molecules. These cell-penetrating peptides, generally categorized as amphipathic (meaning having both a polar and a nonpolar end) or cationic depending on their sequence, provide a non-invasive delivery technology for macromolecules. PTDs also often are referred to as “Trojan peptides”, “membrane translocating sequences”, or “cell permeable proteins” (CPPs). PTDs also may be used to assist novel HSP27 kinase inhibitors to penetrate cell membranes (see U.S. application Ser. No. 11/972,459, entitled “Polypeptic Inhibitors of HSP27 Kinase and Uses Thereof,” filed Jan. 10, 2008, and Ser. No. 12/188,109, entitled “Kinase Inhibitors and Uses Thereof,” filed Aug. 7, 2008, incorporated by reference in their entirety herein).
3.4.1. Viral PTD Containing Proteins
The first proteins to be described as having transduction properties were of viral origin. These proteins still are the most commonly accepted models for PTD action. The HIV-1 Transactivator of Transcription (TAT) and HSV-1 VP 22 protein are the best characterized viral PTD containing proteins.
TAT (HIV-1 trans-activator gene product) is an 86-amino acid polypeptide, which acts as a powerful transcription factor of the integrated HIV-1 genome. TAT acts on the viral genome stimulating viral replication in latently infected cells. The translocation properties of the TAT protein enable it to activate quiescent infected cells and it may be involved in priming of uninfected cells for subsequent infection by regulating many cellular genes, including cytokines. The minimal PTD of TAT is the 9 amino acid protein sequence RKKRRQRRR (TAT49-57) [SEQ ID NO: 4]. Studies utilizing a longer fragment of TAT demonstrated successful transduction of fusion proteins up to 120 kDa. The addition of multiple TAT-PTDs as well as synthetic TAT derivatives have been demonstrated to mediate membrane translocation. TAT PTD containing fusion proteins have been used as therapeutic moieties in experiments involving cancer, transporting a death-protein into cells, and disease models of neurodegenerative disorders.
VP22 is the HSV-1 tegument protein, a structural part of the HSV virion. VP22 is capable of receptor independent translocation and accumulates in the nucleus. This property of VP22 classifies the protein as a PTD containing peptide. Fusion proteins comprising full length VP22 have been translocated efficiently across the plasma membrane.
3.4.2. Homeoproteins with Intercellular Translocation Properties
Homeoproteins are highly conserved, transactivating transcription factors involved in morphological processes. They bind to DNA through a specific sequence of 60 amino acids. The DNA-binding homeodomain is the most highly conserved sequence of the homeoprotein. Several homeoproteins have been described to exhibit PTD-like activity; they are capable of efficient translocation across cell membranes in an energy-independent and endocytosis-independent manner without cell type specificity.
The Antennapedia protein (Antp) is a trans-activating factor capable of translocation across cell membranes; the minimal sequence capable of translocation is a 16 amino acid peptide corresponding to the third helix of the protein's homeodomain (HD). The internalization of this helix occurs at 4° C., suggesting that this process is not endocytosis dependent. Peptides up to 100 amino acids produced as fusion proteins with AntpHD penetrate cell membranes. Other homeodomains capable of translocation include Fushi tarazu (Ftz) and Engrailed (En) homeodomain. Many homeodomains share a highly conserved third helix.
3.4.3. Synthetic PTDs
Several PTD peptides have been synthesized. Many of these synthetic peptides are based on existing and well documented peptides, while others are selected for their basic residues and/or positive charge, which generally are believed to be crucial for PTD function. Synthetic peptides include, but are not limited to, PTD-4 (YARAAARQARA) [SEQ ID NO: 5]; PTD-5 (RRQRRTSKLMKR) [SEQ ID NO: 6]; MST-1 (AAVLLPVLLAAR) [SEQ ID NO: 7]; L-R9 (RRRRRRRRR) [SEQ ID NO: 8]; and Peptide 2 (SGWFRRWKK) [SEQ ID NO: 9].
3.4.4. Human PTDs
Human PTDs may circumvent potential immunogenicity issues upon introduction into a human patient. Peptides with PTD sequences include: Hoxa-5, Hox-A4, Hox-B5, Hox-B6, Hox-B7, HOX-D3, GAX, MOX-2, and FtzPTD. These proteins all share the sequence found in AntpPTD (RQIKIWFQNRRMKWKK) [SEQ ID NO: 10]. Other PTDs include Islet-1, interleukin-1β, tumor necrosis factor, and the hydrophobic sequence from Kaposi-fibroblast growth factor or FGF-4) signal peptide, which is capable of energy-, receptor-, and endocytosis-independent translocation. Unconfirmed PTDs include members of the Fibroblast Growth Factor (FGF) family.
4. Disorders: Inflammatory Disorders
The term “inflammation” as used herein refers to the physiologic process by which vascularized tissues respond to injury. See, e.g., FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053, incorporated herein by reference. During the inflammatory process, cells involved in detoxification and repair are mobilized to the compromised site by inflammatory mediators. Inflammation is often characterized by a strong infiltration of leukocytes at the site of inflammation, particularly neutrophils (polymorphonuclear cells). These cells promote tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue. Traditionally, inflammation has been divided into acute and chronic responses.
The term “acute inflammation” as used herein refers to the rapid, short-lived (minutes to days), relatively uniform response to acute injury characterized by accumulations of fluid, plasma proteins, and neutrophilic leukocytes. In acute inflammation, removal of the stimulus halts the recruitment of monocytes (which become macrophages under appropriate activation) into the inflamed tissue, and existing macrophages exit the tissue via lymphatics. Examples of injurious agents that cause acute inflammation include, but are not limited to, pathogens (e.g., bacteria, viruses, parasites), foreign bodies from exogenous (e.g. asbestos) or endogenous (e.g., urate crystals, immune complexes), sources, and physical (e.g., burns) or chemical (e.g., caustics) agents.
The term “chronic inflammation” as used herein refers to inflammation that is of longer duration and which has a vague and indefinite termination. Chronic inflammation takes over when acute inflammation persists, either through incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring in the same location. Chronic inflammation, which includes the influx of lymphocytes and macrophages and fibroblast growth, may result in tissue scarring at sites of prolonged or repeated inflammatory activity. In chronic inflammation, existing macrophages are tethered in place, and proliferation of macrophages is stimulated.
Regardless of the initiating agent, the physiologic changes accompanying acute inflammation encompass four main features: (1) vasodilation, which results in a net increase in blood flow, is one of the earliest physical responses to acute tissue injury; (2) in response to inflammatory stimuli, endothelial cells lining the venules contract, widening the intracellular junctions to produce gaps, leading to increased vascular permeability which permits leakage of plasma proteins and blood cells out of blood vessels; (3) inflammation often is characterized by a strong infiltration of leukocytes at the site of inflammation, particularly neutrophils (polymorphonuclear cells). These cells promote tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue; and (4) fever, produced by pyrogens released from leukocytes in response to specific stimuli.
During the inflammatory process, soluble inflammatory mediators of the inflammatory response work together with cellular components in a systemic fashion in the attempt to contain and eliminate the agents causing physical distress. The term “inflammatory mediators” as used herein refers to the molecular mediators of the inflammatory process. These soluble, diffusible molecules act both locally at the site of tissue damage and infection and at more distant sites. Some inflammatory mediators are activated by the inflammatory process, while others are synthesized and/or released from cellular sources in response to acute inflammation or by other soluble inflammatory mediators. Examples of inflammatory mediators of the inflammatory response include, but are not limited to, plasma proteases, complement, kinins, clotting and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet-activating factor (PAF), peptides and amines, including, but not limited to, histamine, serotonin, and neuropeptides, proinflammatory cytokines, including, but not limited to, interleukin-1, interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF), interferon-gamma, and interleukin 12 (IL-12).
Several disorders associated with inflammation underlie a variety of diseases. These include, but are not limited to, asthma, autoimmune diseases, chronic prostatitis, glomerulonephritis, inflammatory bowel disease (IBD), pelvic inflammatory disease (PID), reperfusion injury, rheumatoid arthritis, vasculitis and hypersensitivities.
Asthma
Asthma is a chronic disease involving the respiratory system in which the airways may constrict sporadically, become inflamed, and are lined with excessive amounts of mucus, often in response to one or more triggers. These triggers may include, but are not limited to, exposure to an environmental stimulants such as, but not limited to, allergens, smoke, cold or warm air, perfume, pet dander, moist air, exercise or exertion or emotional distress. The airway narrowing presents symptoms such as, but not limited to, wheezing, shortness of breath, chest tightness, coughing, dyspnea, and stridor. Elevated serum levels of IL-6 in subjects with asthma compared with normal control subjects have been implicated in the pathophysiology of bronchial asthma. Yokoyama, A. et al., Am. J. Respir. Crit. Care Med. 151(5): 1354-58 (1995). Studies also suggested, based on the observation that significant levels of TNF-α and IL-6 were detected in bronchoalveolar lavage fluid (BALF) of asthma patients, while levels of IL-1β levels in patients BALF of patients with asymptomatic asthma, activation of alveolar macrophages and T cells (Broide, D. H., et al. J. Allergy Clin. Immunol. 89(5):958-67, 1992).
Autoimmune Diseases
Ankylosing spondylitis (AS, Bechterew's disease, Bechterew syndrome, Marie Strumpell disease) is a chronic, inflammatory arthritis and autoimmune disease. It mainly affects joints in the spine and the sacroilium in the pelvis, causing eventual fusion of the spine. Studies have reported that TNF-α and IL-6 are increased in AS patients (while IL-1β levels are not) and that IL-6 is closely correlated with the activity of the disease (Gratacos, J., et al. Br. J. Rheumatol. 33(10):927-931. 1994). Symptoms of AS include, but are not limited to, chronic pain and stiffness in the lower part of the spine or sometimes the entire spine, often with pain referred to one or other buttock or the back of the thigh from the sacroiliac joint, inflammation of the eye (iridocyclitis, uveitis) causing redness, eye pain, vision loss, floaters, photophobia, fatigue, nausea, aortitis, apical lung fibrosis, and ectasia of the sacral nerve root sheaths.
Type 1 diabetes is an autoimmune disease whereby the islet cells of the pancreas come under attack from T-cells, which renders the body incapable of producing insulin. It has been reported that β-cell destructive insulitis is associated with increased expression of IL-1 and TNF-α. Further, transgenic expression of cytokines in pancreatic islet β-cells of non-diabetes-prone mice and non-obese diabetic (NOD) mice has suggested pathogenic roles for IFNα, IFNγ, IL-2 and IL-10 in insulin-dependent diabetes mellitus (IDDM) development and protective roles for IL-4, IL-6 and TNF-α(Rabinovitch, A. Diabetes Metab. Rev. 14:129-151, 1998). Symptoms of type 1 diabetes include, but are not limited to, polyuria, polydispia and weight loss.
Guilliamé-Barre syndrome is an acute inflammatory demyelinating polyneuropathy (an autoimmune disorder affecting the peripheral nervous system). It frequently is severe, and usually exhibits as an ascending paralysis noted by weakness in the legs that spreads to the upper limbs and the face along with complete loss of deep tendon reflexes. Studies have reported that the differential expression of IL-1β, IL-6, and TNF-α in an animal model of the disease argues for a major role of these cytokines (Zhu, J., et al. Clin. Immunol. Immunopathol. 84(1):85-94. 1997). Symptoms of Guilliamé-Bane syndrome include, but are not limited to, symmetrical weakness which usually affects the lower limbs first, and rapidly progresses in an ascending fashion, “rubbery legs” with or without dysesthesias, bulbar weakness (oropharyngeal dysphagia), respiratory difficulties, facial weakness, sensory loss (proprioception), wide fluctuations in blood pressure, orthostatic hypotension, and cardiac arrhythmias.
Lupus is a chronic autoimmune connective tissue disease, affecting any part of the body, causing inflammation and tissue damage. Lupus most often harms the heart, joints, skin, lungs, blood vessels, liver, kidneys, and nervous system. Studies have shown that IL-6 and TNF-α actively are synthesized in the kidneys of patients with lupus nephritis (Herrera-Esparza, R., et al. Lupus. 7(3):154-158, 1998). Additional studies have reported that expression of TNF-α and IL-1β are elevated in animal models of lupus nephritis (Boswell, J., et al. J. Immunol. 141(9):3050-3054, 1988). Symptoms of lupus include, but are not limited to, fatigue, fever, weight gain or loss, joint pain, stiffness, swelling, malar rash on the face, skin lesions, mouth sores, alopecia, shortness of breath, chest pain, dry eyes and Raynaud's phenomenon.
Multiple sclerosis (MS) is an autoimmune disease that affects the myelinated neurons of the brain and spinal cord. MS is caused by damage to the myelin sheath; nerve impulses are slowed or stopped when this covering is damaged. Studies have reported increased expression of TNF-α in MS cases (Cannella, D., et al. Ann. Neurol. 37(4):424-435, 2004) and of IL-6 in lesions from MS patients (Lock, C., et al. Nature Medicine. 8:500-508, 2002). Symptoms of multiple sclerosis include, but are not limited to, loss of balance, muscle spasms, numbness or abnormal sensation in any area, problems moving arms or legs and walking, tremor in one or more arms or legs, constipation, stool leakage, incontinence, double vision, eye discomfort, facial pain, and hearing loss.
Psoriasis is a chronic, non-contagious autoimmune disease that affects the skin and joints. It commonly causes red, scaly patches to appear on the skin. These psoriatic plaques are areas of inflammation and excessive skin production. Skin rapidly accumulates at these sites and takes on a silvery-white appearance. Plaques can affect any area, including the elbow, the knee, the scalp, and the genitals. Studies have reported elevated levels of TNF-α, IL-1β and IL-6 in psoriasis patients (Mizutani, H., et al. J. Dermatol. Sci. 14(2):145-153. 1997).
Scleroderma is a widespread connective tissue disease that involves changes in the skin, blood vessels, muscles and internal organs. Studies have reported that IL-6 was detected more frequently in sera from scleroderma patients than in sera from controls, and that TNF-α was detected at the same levels in both patient groups, while IL-1β was not detected from either group (Needleman, B. W., et al. Arthritis Rheum. 35(1):67-72, 1992). Skin symptoms include, but are not limited to, blanching, blueness, or redness of fingers and toes in response to heat and cold (Raynaud's phenomenon), hair loss, skin hardness, skin is abnormally dark or light, skin thickening and shiny hands and forearms, and ulcerations on fingertips or toes. Bone and muscle symptoms include, but are not limited to, joint pain, numbness and pain in the feet, pain, stiffness and swelling of fingers and joints, wrist pain. Additional symptoms include, but are not limited to, constipation, diarrhea, dry cough, wheezing, and difficulty swallowing.
Sjogren's disease (Mikulicz disease, Sicca syndrome) is an autoimmune disorder in which immune cells attack and destroy the exocrine glands that produce tears and saliva. Studies have shown that IL-1β, IL-6 and TNF-α levels are significantly different between patients with Sjogren's disease and normal healthy controls (Szodoray, P., et al. Scand. J. Immunol. 59(6):592-599). Symptoms of Sjogren's disease include, but are not limited to, dry mouth, dry eyes, skin dryness, nose dryness, and vaginal dryness.
Glomerulonephritis
Glomerulonephritis (glomerular neprhitis, GN) is a renal disease characterized by inflammation of the small blood vessels (glomeruli) of the kidney. Studies have reported that the inflammatory cytokines IL-1 and TNF-α each play a role in the immune/inflammatory process in glomerulonephritis and that blocking their action reduces disease (Atkins, Nephrology. 7(s1):S2-S6, 2007); Johnson, R. J., Nephron. 73(4):506-514, 1996). Additional studies have reported that IL-6 also plays a role in glomerulonephritis (Takemura, T., et al. Virchows Archiv. 424(5):459-464, 1994). Symptoms of glomerulonephritis include, but are not limited to, edema, high blood pressure, and the presence of red blood cells in the urine.
Urologic Chronic Pelvic Pain
Urologic chronic pelvic pain syndromes refers to pain syndromes associated with the bladder (i.e., interstitial cystitis (IC), painful bladder syndrome (PBS)) and the prostate gland (chronic prostatitis (CP), chronic pelvic pain syndrome (CPPS)). Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is characterized by pelvic or perineal pain without evidence of urinary tract infection, lasting longer than 3 months. Studies have reported that levels of IL-1β, TNF-α and IL-6 were elevated significantly in groups having inflammatory and non-inflammatory CPPS compared with a control group (Orhan, I., et al. Int. J. Urol. 8(9):495-9, 2001; Alexander, R. B., et al. Urology. 52(5):744-749, 1998; Jang, T. L., and Schaeffer, A. J., World J. Urol. 21(2):95-99, 2003). Symptoms of these syndromes may wax and wane. Pain may range from mild discomfort to debilitating, and may radiate from the back and rectum, making sitting difficult. Dysuria (difficult or painful urination), arthralgia (pain in a joint), myalgia (pain in the muscles), unexplained fatigue, abdominal pain, constant burning pain in the penis, and frequency may all be present. Frequent urination and increased urgency may suggest interstitial cystitis (inflammation centered in the bladder rather than prostate). Ejaculation may be painful, as the prostate contracts during emission of semen, although nerve- and muscle-mediated post-ejaculatory pain is more common. Some patients report low libido, sexual dysfunction and erectile difficulties. Pain after ejaculation is a very specific complaint that distinguishes CP/CPPS from men with benign prostatic hyperplasia (BPH) or normal men.
Inflammatory Bowel Disease (IBD)
The term “Inflammatory Bowel Disease (IBD)” refers to a group of inflammatory conditions of the large intestine and small intestine. IBDs include Crohn's disease (CD) and ulcerative colitis (UC). Other forms of IBD include collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, infective colitis and indeterminate colitis. Many of these disorders may present the symptoms of abdominal pain, vomiting, diarrhea, hematochezia (bright red blood in stools), weight loss and various associated complaints or diseases like arthritis, pyoderma gangrenosum, and primary sclerosing cholangitis. Diagnosis is generally by colonoscopy with biopsy of pathological lesions. Studies have reported that cytokines such as IL-6, IL-1 and TNF-α play a central role in the modulation of the intestinal immune system and that the mucosal and systemic concentrations of many pro- and antiinflammatory cytokines are elevated in IBD (Rogler, G., and Andus, T. World J. Surg. 22(4):382-9, 1998). Studies also have shown elevated levels of TNF-α, IL-1β and IL-6 in Crohn's patients and that the concentrations of IL-1β and IL-6 in the supernatant fluid of biopsy cultures are positively correlated with the degree of tissue involvement measured by both endoscopic and histological grading (Reimund, J., et al. Gut. 39:684-689, 1996).
Pelvic Inflammatory Disease (PID)
Pelvic inflammatory disease (PID) refers to inflammation of the female uterus, fallopian tubes, and/or ovaries as it progresses to scar formation with adhesions to nearby tissues and organs. PID may lead to tissue necrosis and abscess formation. Studies have reported that IL-1β, IL-6 and TNF-α are significantly elevated in PID patients before antibiotic treatment (as compared to after treatment) and that these cytokines may play an important role in the pathogenesis of PID (Lee, S. A., et al. Clin. Chem. Lab. Med. 46(7):997-1003, 2008).
Reperfusion Injury
Reperfusion injury refers to damage to tissue caused when blood supply returns to the tissue after a period of ischemia. The absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function. Studies have reported that IL-6 prevents the liver against warm ischemia/reperfusion injury through down regulation of TNF-α (Camargo, C., et al. Hepatology. 26(6):1513-1520, 2003). Symptoms include, but are not limited to, elevated white blood cell levels, apoptosis, and free radical accumulation.
Rheumatoid Arthritis (RA)
Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disorder that most commonly causes inflammation and tissue damage in joints (arthritis) and tendon sheaths, together with anemia. RA also may produce diffuse inflammation in the lungs, pericardium, pleura, and the sclera of the eye, and also nodular lesions, most common in subcutaneous tissue under the skin. RA may be a disabling and painful condition, which may lead to substantial loss of functioning and mobility. Studies have reported that levels of IL-1β, IL-6 and TNF-α are elevated in the serum of RA and juvenile arthritis patients (Ziolkowska, M., et al. J. Immunol. 164:2832-2838, 2000). Symptoms of RA may manifest in the joints (swelling, pain, tenderness, a sensation of localized warmth, stiffness and restricted movement); skin (rheumatoid nodule); lungs (fibrosis, Caplan's syndrome, pleural effusions); kidneys (renal anlyoidosis); heart and blood vessels (atherosclerosis, myocardial infarction, stroke); and eyes (episcleritis, keratoconjunctivitis sicca).
Vasculitis
“Vasculitis” refers to a disorder characterized by inflammatory destruction of blood vessels (arteries and veins). Studies have reported that TNF-α, IL-1, and IL-6 are potential biological targets for the treatment of systemic vasculitis (Levine, S. M., and Stone, J. H. Best Prac. Res. Clin. Rheumatol. 15(2):315-333, 2001. Symptoms of vasculitis usually are systemic with single or multiorgan dysfunction. These symptoms may include fatigue, weakness, fever, arthralgias, abdominal pain, hypertension, renal insufficiency, and neurologic dysfunction. Additional symptoms may include mononeuritis multiplex, palpable purpura (purple patches on the skin) and pulmonary-renal syndrome. Hypersensitivity vasculitis (HSV) is a secondary vasculitis due to an immune response to exogenous substances. Studies have reported that serum IL-6 and TNF-α is significantly higher in active HSV patients than in a healthy control group (Nalbant, S., et al. Rheumatol. Int. 22(6):244-248, 2002).
5. Disorders: Fibrosis
Fibrosis is the formation or development of excess fibrous connective tissue in an organ or tissue as a result of injury or inflammation of a part, or of interference with its blood supply. It may be a consequence of the normal healing response leading to a scar, or it may be an abnormal, reactive process.
There are several types of fibrosis including, but not limited to, cystic fibrosis of the pancreas and lungs, injection fibrosis, endomyocardial fibrosis, idiopathic pulmonary fibrosis of the lung, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, and nephrogenic systemic fibrosis.
Cystic fibrosis (CF, mucovidosis, mucovisidosis) is an inherited autosomal recessive disorder. It is one of the most common fatal genetic disorders in the United States, affecting about 30,000 individuals, and is most prevalent in the Caucasian population, occurring in one of every 3,300 live births. The gene involved in cystic fibrosis, which was identified in 1989, codes for a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR normally is expressed by exocrine epithelia throughout the body and regulates the movement of chloride ions, bicarbonate ions and glutathione into and out of cells. In cystic fibrosis patients, mutations in the CFTR gene lead to alterations or total loss of CFTR protein function, resulting in defects in osmolarity, pH and redox properties of exocrine secretions. In the lungs, CF manifests itself by the presence of a thick mucus secretion which clogs the airways. In other exocrine organs, such as the sweat glands, CF may not manifest itself by an obstructive phenotype, but rather by abnormal salt composition of the secretions (hence the clinical sweat osmolarity test to detect CF patients). The predominant cause of illness and death in cystic fibrosis patients is progressive lung disease. The thickness of CF mucus, which blocks the airway passages, is believed to stem from abnormalities in osmolarity of secretions, as well as from the presence of massive amounts of DNA, actin, proteases and prooxidative enzymes originating from a subset of inflammatory cells, called neutrophils. Indeed, CF lung disease is characterized by early, hyperactive neutrophil-mediated inflammatory reactions to both viral and bacterial pathogens. The hyperinflammatory syndrome of CF lungs has several underpinnings, among which an imbalance between pro-inflammatory chemokines, chiefly IL-8, and anti-inflammatory cytokines, chiefly IL-10, has been reported to play a major role. See Chmiel et al. Clin Rev Allergy Immunol. 3(1):5-27 (2002). Studies have reported that levels of TNF-α, IL-6 and IL-1β were higher in the bronchoaveolar lavage fluid of cystic fibrosis patients, than in healthy control bronchoaveolar lavage fluid (Bondfield, T. L., et al. Am. J. Resp. Crit. Care Med. 152(1):2111-2118, 1995).
Injection fibrosis (IF) is a complication of intramuscular injection often occurring in the quadriceps, triceps and gluteal muscles of infants and children in which subjects are unable to fully flex the affected muscle. It typically is painless, but progressive. Studies have reported that the glycoprotein osteopontin (OPN) plays a role in tissue remodeling (Liaw, L., et al. J. Clin. Invest, 101(7):1469-1478, 1998) and that this proinflammatory mediator induces IL-1β up-regulation in human monocytes and an accompanying enhanced production of TNF-α and IL-6 (Naldini, A., et al. J. Immunol. 177:4267-4270, 2006; Weber, G. F., and Cantor, H. Cytokine Growth Factor Reviews. 7(3):241-248, 1996).
Endomyocardial disease (hyperosinophilic syndrome (HS)) is a disease process characterized by a persistently elevated eosinophil count (≧1500 eosinophils/mm3) in the blood. HS affects simultaneously affects many organs, Studies have reported that IL-1β, IL-6 and TNF-α are expressed at high levels in viral-induced myocarditis patients (Satoh, M., et al. Virchows Archiv. 427(5):503-509, 1996). Symptoms may include cardiomyopathy, skin lesions, thromboembolic disease, pulmonary disease, neuropathy, hepatosplenomegaly (coincident enlargement of the liver and spleen), and reduced ventricular size. Treatment may include utilizing corticosteroids to reduce eosinophil levels.
Idiopathic pulmonary fibrosis (IPF, cryptogenic fibrosing alveolitis) is a chronic progressive interstitial lung disease of unknown cause. It is associated with a histological pattern of usual interstitial pneumonia and may be characterized by abnormal and excessive deposition of fibrotic tissue in the pulmonary interstitium with minimal associated inflammation. Studies have reported significant increases in TNF-α and IL-6 release in patients with idiopathic pulmonary fibrosis (IPF) (Zhang, Y., et al. J. Immunol. 150(9):4188-4196, 1993), which has been attributed to the level of expression of IL-1β (Kolb, M., et al. J. Clin. Invest, 107(12):1529-1536, 2001). Symptoms include dyspnea (difficulty breathing), but also include nonproductive cough, clubbing (a disfigurement of the fingers), and crackles (crackling sound in lungs during inhalation).
Mediastinal fibrosis (MF) is characterized by invasive, calcified fibrosis centered on lymph nodes that block major vessels and airways. MF is a late complication of histoplasmosis. Studies in murine models of fibrosis have reported that IL-10 and TNF-α are elevated significantly (Ebrahimi, B., et al. Am. J. Pathol. 158:2117-2125, 2001).
Myelofibrosis (myeloid metaplasia, chronic idiopathic myelofibrosis, primary myelofibrosis) is a disorder of the bone marrow in which the marrow undergoes fibrosis. Myelofibrosis leads to progressive bone marrow failure. The mean survival is five years and causes of death include infection, bleeding, organ failure, portal hypertension, and leukemic transformation. It has been reported that TNF-α and IL-6 levels are elevated in animal models of viral-induced myelofibrosis (Bousse-Kerdiles, M., et al. Ann. Hematol. 78:434-444, 1999).
Retroperitoneal fibrosis (Ormond's disease) is a disease featuring the proliferation of fibrous tissue in the retroperitoneum. The retroperitoneum is the body compartment containing the kidneys, aorta, renal tract, and other structures. It has been reported that IL-1, IL-6 and TNF-α have key roles in the pathogenesis of retroperitoneal fibrosis (Demko, T., et al, J. Am. Soc. Nephrol. 8:684-688, 1997). Symptoms of retroperitoneal fibrosis may include, but are not limited to, lower back pain, renal failure, hypertension, and deep vein thrombosis.
Nephrogenic systemic fibrosis (NSF, nephrogenic fibrosing dermopathy) involves fibrosis of the skin, joints, eyes and internal organs. NSF may be associated with exposure to gadolinium. Patients develop large areas of hardened skin with fibrotic nodules and plaques. Flexion contractures with an accompanying limitation of range of motion also may occur. NSF shows a proliferation of dermal fibroblasts and dendritic cells, thickened collagen bundles, increased elastic fibers, and deposits of mucin. Some reports have suggested that a proinflammatory state provides a predisposing factor for causing nephrogenic systemic fibrosis (Saxena, S., et al. Int. Urol. Nephrol. 40:715-724, 2008), and that the level of TNF-α is elevated in animal models of nephrogenic systemic fibrosis (Steger-Hartmann, T., et al. Exper. Tox. Pathol. 61(6):537-552, 2009).
6. Disorders: Endothelial Cell Dysfunction
Endothelial cell dysfunction (endothelial dysfunction) refers to a physiological dysfunction of normal biochemical processes carried out by the endothelium, such as mediation of coagulation, of platelet adhesion, of immune function, and of control of volume and electrolyte content of the intravascular and extravascular spaces. Endothelial dysfunction may result from disease processes, such as, for example, septic shock, hypertension, hypercholesterolaemia, and diabetes as well as from environmental factors, such as from smoking tobacco products. Studies have reported that under the influence of cytokines, such as IL-6, IL-1β, and TNF-α, the endothelium-dependent dilation can be impaired and the endothelium may lose its ability to respond to circulating hormones or autacoids. This effect may favor a predisposition to vessel spasm, thrombosis or atherogenesis (Vila, E. and Salaices, M. Am. J. Physiol, Heart Circ. Physiol. 288:H1016-H1021, 2005). In addition, studies have suggested that the overexpression of IL-6, as regulated by IL-1β and TNF-α, has an important role in endothelial cell dysfunction (Korman, K. et al. J. Perio. Res. 34(7):353-357 (2006); Libby, P., et al. Circulation. 86 (6 Suppl): III47-52 (1992)).
Endothelial dysfunction may be characterized by the inability of arteries and arterioles to dilate fully in response to an appropriate stimulus. For example, dysfunctional endothelial cells (having reduced vasodilation) are unable to produce nitric oxide (NO) to the same extent as healthy endothelial cells. This difference is detectable by a variety of methods including iontophoresis of acetylcholine, intra-arterial administration of various vasoactive agents, localized heating of the skin and temporary arterial occlusion by inflating a blood pressure cuff to high pressures. Testing also may take place in the coronary arteries themselves, however this invasive procedure normally is not conducted unless there is a clinical reason for intracoronary catheterization. These techniques are thought to stimulate the endothelium to release NO which diffuses into the surrounding vascular smooth muscle causing vasodilation.
Systems that allow for delivery of biologically active recombinant proteins and peptide therapeutics have been the subject of numerous studies. Systems for localized delivery of therapeutic agents allow for the biological effects of such agents to achieve greater efficacy.
Challenges persist to deliver recombinant proteins to desired targets in vivo. Despite developments in the area of protein transduction peptides, the classical delivery methods of protein-coding genes via adeno-associated virus, adenovirus, lentivirus, herpes virus vectors, and plasmid expression vectors remain the preferred choice for expression of proteins.
Viral vector-mediated gene expression is considered the most efficient and reliable approach for expressing functional proteins de novo in mitotically active or postmitotically blocked cell types due to the natural ability of such vectors to deliver the specific genes to permissive cells. Nonetheless, viral vectors invariably are required in large doses to achieve therapeutic expression levels of intended proteins. Moreover, viral vectors may integrate with the host chromatin material. These properties exert long term effects on host genetic systems, and therefore, safety remains a serious concern for their ultimate clinical application.
An alternative, safer, approach is to produce recombinant proteins exogenously and then deliver them systemically or by localized injections into the target organs. However, the delivery and bioavailability of recombinant proteins into cells or tissues need further improvements. Although several studies have suggested the potential of PTD in drug discovery and transduction of proteins up to 120 kDa into different cells, questions about potency of PTD mediated protein transduction still remain unsolved. Indeed, some studies have demonstrated failures in PTD-mediated fusion protein transduction in vitro/in vivo as well as an inability to induce an immune response. Further, some studies have shown that intracellular expression of PTD fusion proteins or other non-secretory proteins may not achieve the same biodistribution as recombinant protein, and entry of PTD through the blood-brain barrier remains elusive.
The described invention provides therapeutic inhibitor peptides for the inhibition of kinases, uses of a class of peptides that include therapeutic domains and protein transduction domains as inhibitors of kinase activity, and uses of PTDs as therapeutic agents for a variety of disorders.